The present invention relates to a method for preparing liquid-core microcapsules for cell cultures, and in particular to a method for preparing liquid-core microcapsules for cell cultures, which uses a hardening solution containing CaCl.sub.2 and polyethyleneimine to harden gel-core beads before coating them with polylysine solution.
When culturing high-density animal cells, the use of liquid-core microcapsules has many advantages, for example, low cost, ease of culturing and high suitability for culturing various animal cells. Due to the strict requirements on the growing conditions for animal cell cultures, when the microencapsulation technique is used for culturing animal cells, the materials selected for the microcapsules and the method of encapsulation are usually more stringent than the fixing conditions of the cells.
T. Yoshioka et al disclose a process for producing liquid-core microcapsules, which involves adding a polyelectrolyte compound dropwisely to another polyelectrolyte compound of opposite charge (Biotechnol. Bioeng. Vol. 35, pp 66, 1990).
M. C. Bane et al disclose microcapsules made of a new synthetic polymer (Biotechnol. Bioeng. Vol. 9, pp 468, 1991). S. Iijima et al disclose a method for producing gel-core microcapsules by using interfacial polymerization wherein the gel-core contains alginate and polyurea (Appl. Microbiol. Biotechnol. Vol. 28, pp 572, 1988).
In the high-density culturing of animal cells, using liquid-core microcapsules can provide larger growing space for the animal cells encapsulated therein as compared to gel-core microcapsules, and thereby provide higher production. However, as some liquid-core microcapsules are fragile, when the culturing is conducted in a large scale culture device, they are easily ruptured due to mechanical collisions caused by necessary stirring and aeration, causing leakage of the cells and accordingly affecting the whole manufacturing process thereof. E. R. Mckillip et al have reported the problems encountered when cells are mass-cultured by using liquid-core microcapsules (Bio./Technol., Vol. 9, pp 805, 1991).
Heretofore liquid-core microcapsules are produced by mixing a 1-3% alginate solution as core material with cultured cells to form a mixture, dropping the mixture into a 1-3% CaCl.sub.2 solution (hardening solution) to gel the mixture to form gel-core beads, placing the gel-core beads in a solution containing 0.1% of polylysine or polyethyleneimine to coat the beads and form a membrane thereon, followed by dissolving the calcium ion in the gel cores with sodium citrate. The resulting liquid-core microcapsules, when used in culture of animal cells, are able to produce 10.sup.8 cells per ml. However, they have the following disadvantages:
(1) The lifetime of the liquid-core microcapsules is only 2 weeks when they are used for animal cell culture due to their weak mechanical strength. PA1 (2) In accordance with the conventional processes, the operating time for each step influences the quality of the final microcapsules greatly. For example, assuming the coating time of polylysine is set at 3 minutes, increasing or reducing the coating time will result in the instability of the resulting liquid-core microcapsules. Therefore, according to the above-mentioned conventional manufacturing process, when mass production is desired, it is difficult to obtain liquid-core microcapsules having controlled quality. PA1 (3) If cells capable of producing high molecular weight proteins (MW.gtoreq.160 Kd), for example, monoclonal antibodies, are encapsulated in liquid-core microcapsules prepared by the above conventional method, as the produced protein can not permeate the microcapsule membrane and therefore can not be released thereform, the continuous production of the cells will be hindered.
Gel-core microcapsules made of alginate and polyurea as proposed by S. Iijima et al can overcome a part of the above disadvantages, however, they have the disadvantage that the cell density is low, and can not reach the value of 10.sup.8 cells per ml.